Abstract
A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.
Highlights
A full-length rat androgen receptor cDNA was used induced nuclear transcription factors, which includes recepto produce a recombinant baculovirus (AcrAR) by ho- tors for steroid and thyroid hormones, vitamin D, and retimologous recombination
Cd2+ions were capable of restoring the work, we have introduced rat androgen receptor cDNA
Construction of the Recombinant Baculouirus-A full-length third of the total protein and, as expected from the way by rat Androgen receptor (AR) cDNAwas insertedintothe baculovirus transfer which the recombinant transfer vector was constructed, no plasmid pVL1393 usingthe EcoRI andPstIsites of the polyhedrin protein was expressed in these cells (Fig. 2 A, lane multiple cloningsequence (Fig. 1).The resulting recombinant 3 ) . transfer vector contains32 nucleotides of the The above data provided strong circumstantialevidence for 5”untranslated region, the entire protein-codingsequence of the expression of a full-length AR proteinininsect cells 2706 n t for 902 amino acidresidues, and 87 nt of the 3’- infected with the recombinant virusDNA
Summary
PURIFICATIONOF THERECEPTOR AND RENATURATION OF ITS STEROID- AND DNA-BINDING FUNCTIONS*. Zn2+ions were essential for the purified receptor to refold into a specific DNA-binding form functional similarityof these proteins to those present under physiological conditions in mammalian cells. Production of AR Antibodies-In addition to two antisera against Solubilization of the Recombinant Androgen Receptor-Insoluble synthetic peptides corresponding to residues 479-495 (peptide 1)and pellets washed as above were suspended in a small volume of NTE. Purification of the Recombinant Androgen Receptor by Gel Filtration-The AR protein present inthe 6 M GdnHC1-solubilizedSf9 cell kDa polyhedrin protein (see below)a, nd appeared to represent pure strains of recombinant AR-producing viruses One of these strains (designatedAcrAR) was selected for subsequent pellet was purified by gel filtration on a Sephacryl S-400 HR(Phar- experiments. Cells infected with the wild-type baculoviruscontained a largeamount of the 30-kDa tron microscopy as previously described [33]
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